The invention relates to using bifunctional two-domain binding molecules to recruit therapeutic agent to a solid tissue site, wherein the bifunctional binding molecules are administered first, and once the binding molecules reach maximum concentration in the extravascular space, a remover substance is administered to aid in clearing the binding molecules in the blood circulation and extravascular space, and thereafter, the therapeutic agent is administered.
Much research and experimentation has been done on how to deliver therapeutic and imaging agents to solid tissue sites in vivo. Such site-specific delivery has often been attempted with monoclonal antibodies (xe2x80x9cmAbsxe2x80x9d) conjugated with the therapeutic or imaging agents. These immunoconjugates are often called xe2x80x9cmagic bulletsxe2x80x9d, because of their ability to specifically target diseased or tumorous sites in vivo.
Immunotoxins, which are immunoconjugates in which mAbs are conjugated with toxic substances, such as plant or bacterial-derived toxins including pseudomonas exotoxin, ribosomal-inactivating proteins, ricin, gelonin, and pokeweed antiviral peptide, have also been extensively studied. Additionally, mAbs conjugated with metal-chelating agents, where the metal-chelating agents can carry radioactive isotopes, have been used for both treating and imaging tumors.
Immunoconjugates, and particularly immunotoxins, have been actively investigated for treatment of tumors both in solid tissue and in other areas. Clinical trials of immunotoxins for removing tumors or decreasing tumor loads have been conducted. Such tests have often been with immunotoxins where the mAb is conjugated with the A chain of ricin or a radioactive isotope. Immunotoxins have also been studied in animal models for eliminating malignant cells in tumors transplanted into the animals.
These studies indicate that immunotoxins are more effective in treating leukemia or lymphoma than solid tumors. One plausible explanation for this difference in efficacy is that malignant cells in blood or lymphoid tissues are more accessible than those in solid tumors. Thus, many malignant cells in a solid tumor come in contact only with insufficient amounts of toxin to kill them. In addition, even where the toxin is in contact with the target cells, only a very small fraction will actually enter the cell and thus, not all cells in a solid tumor will be killed.
It is possible, of course, to increase the total amount of immunotoxin administered, in order to increase that which is in the vicinity of malignant cells and available to kill the cells. However, because of the conjugation with the antibody molecules, much of the immunotoxin is also absorbed and taken up by the reticuloendothelial cells of the body. The toxin will damage or destroy these cells. Specifically, a large proportion of immunotoxin ends up in the phagocytic cells in the liver, where, because of its toxicity, it can damage the liver and its function. Thus, the total amount of toxin which can be administered is severly limited.
An illustration of the problems encountered with immunotoxins is seen in a typical clinical trial. See Parker, S. A. et al. xe2x80x9cTherapeutic Monoclonal Antibodiesxe2x80x9d Ed. by Borrebaeck, C. A. and Larrick, J. W. pp. 127-141 (Stockton Press, New York 1990). Patients with B cell lymphoma were treated with anti-idiotype antibodies coupled with the radioactive isotype 90Yttrium. This therapy proved so toxic that the immunoconjugate had to be administered with excess cold, unlabeled anti-idiotype antibodies. However, the excess cold anti-idiotypes competed with the labeled immunoconjugates for binding to the tumor associated antigen, and thereby inhibited the binding of the immunoconjugates to the tumor cell targets.
Similar drawbacks result where an immunoconjugate which includes a mAb and a radioactive isotype is used for tumor imaging. The immunoconjugate tends to be bound and taken up in phagocytic cells in the liver, spleen, and blood circulation, because the antibody portion of the immunoconjugate is absorbed by these cells. This increases the background xe2x80x9cnoisexe2x80x9d and interferes with tumor imaging, and it can also cause toxic levels of radioactivity in all of these organs.
Several groups have tried to solve the major problem which results when using mAbs coupled with imaging agents, ie., the imaging agent is absorbed in vivo and cleared together with the antibody. One group suggested that instead of coupling the mAbs and the imaging agents, a bispecific antibody, which is not coupled to an imaging agent, should be administered first. The bispecific antibody has one specificity against the tumor being targeted and the other against a chelate conjugated to a peptide. The bispecific antibody distributes between the tumor and the circulation, and at a point when there is a high tumor-to-background ratio, a labeled chelate is administered. The chelate which is not absorbed by the antibody is rapidly excreted by the kidneys, due to its relatively small size. This results in low background noise. See Monoclonal Antibodies in Immunoscintigraphy Ed. by Chatal, J. F., pp. 70-71 (CRC Press, Boca Raton, Fla. 1989).
Another group discussed administering an antibody which slowly diffuses to the target tumor, and then clearing the excess circulating antibody. The clearance is done with an antigen covalently bound to a slowly diffusable serum protein (human transferrin). Thereafter, the imaging tracer is administered as an epitopically derivatized bifunctional chelate which is small and rapidly diffusable, and quickly cleared. Again, this is designed to help reduce background radiation and improve imaging. See Goodwin, D. A. et al., J. Nuc. Med. 29:226-34 (1988). A related paper suggested using bifunctional antibodies such as two Fab"" fragments coupled at the SH groups, where one specificity is for the chelate and the other is for the tumor site antigen. See Goodwin, D. A., J. Nuc. Med. 28:1358-62 (1987).
Another related paper suggested injecting antibody and labeled protein (transferrin) followed by injection of anti-human IgG antibody and anti-transferrin antibody. The second antibody injection helps to clear excess labeled transferrin and reduce the background noise. See Goodwin, D. A. et al., J. Nuc. Med. 9:209-215 (1984).
None of these articles discuss how to clear both the blood vessels and the extravascular space of binding molecules prior to administering the imaging agents, while retaining, attached to the target tissue, as much as possible of the binding molecules. When administering toxins or therapeutic agents, it is even more important to clear binding molecules from the extravascular space (as well as from the blood vessels) so that excess toxin is not bound by the binding molecules in the extravascular space and does not cause damage. Thus, if bispecific binding molecules with one specificity for the target site and one for the toxin/therapeutic agent are administered initially, those molecules which bind to the target site must be retained as much as possible, and those molecules which are unbound and in the circulation or extravascular space should be removed. It is also important that the removal should be accomplished quickly enough so that the binding molecule is not released from the target site before the toxin/therapeutic agent is administered.
A number of factors must be considered in designing an effective method of treating solid tumors using tissue-specific recruiting by a binding molecule of a therapeutic agent. These factors include:
1) the pharmokinetic properties of the binding molecules, therapeutic agents, and other substances used in the method;
2) the clearance routes (reticuloendothelial system versus kidney) of the binding molecules, therapeutic agents and other substances;
3) the diffusion rates of the binding molecules and therapeutic agents in and out from the capillaries;
4) the binding molecules must not be endocytosed by the cells;
5) the on/off times of the binding molecules on the target cells;
6) the affinity of the binding molecules for the therapeutic agents, and the efficiency with which they can recruit the therapeutic agents to the target site;
7) the therapeutic cytotoxins such as ricin A chain, pokeweed antiviral peptide, must enter the target cells to render effects, whereas some other therapeutic substances (and imaging agents) need not enter the target cells to be effective;
8) the immunogenicity and antigenicity of the binding molecules and the therapeutic agents.
These factors make designing an effective method very complex.
The invention includes using bifunctional two-domain binding molecules to recruit a therapeutic agent to a solid tissue target site, where the binding molecules have one specificity for the target site and the other specificity for the therapeutic agent. The therapeutic agent is administered separately, after administering the binding molecules and after administering a remover substance.
The remover is preferably a liposome which is conjugated with antibodies against the binding molecules. The remover cannot diffuse into the extravascular space and is rapidly removed by the phagocytic cells in the liver, spleen and blood circulation. It binds to binding molecules which are in the circulation, which thereby facilitates the clearing of the binding molecules from the circulation. After clearance, there is a concentration difference in binding molecules across the blood vessel wall, and binding molecules in the extravascular space diffuse into the blood vessels.
The remover should be administered as soon as possible after the binding molecules reach a maximum concentration in the vascular space. When administered at such time, substantial amounts of binding molecule have not yet been released from the target tissue site.
The remover is preferably administered at least twice, and the subsequent administrations of the remover are at a time after the binding molecules in the circulation and extravascular space have reached equilibrium. The administration of remover effectively clears the majority of the binding molecule in both the extravascular space and in the circulation, and will therefore further increase the ratio of binding molecules in the target site over binding molecules in the circulation and extravascular space.
The therapeutic agent should be administered after the last administration of the remover, and after the remover has had enough time to clear from the circulation. But the therapeutic agent should be administered before substantial amounts of binding molecules are released from the target site, so that as much of the therapeutic agent as possible will be bound at the target tissue site by the binding molecules.
The bifunctional two-domain binding molecules are preferably two VH-VL single-chain binding molecules which are joined together. They can be joined with a linking peptide, as described in U.S. Pat. Nos. 5,132,405 and 5,091, 513. See, e.g., FIG. 2D. It is possible to design the remover to include anti-idiotype antibodies which recognize one or more of the binding sites of the binding molecules. In a preferred embodiment, however, an antibody conjugated to the remover recognizes an antigenic structure associated with the joining region between the two VH-VL single-chain binding molecules, or the antibody recognizes the linking peptide itself Alternatively, the peptide joining the two VH-VL single-chain binding molecules may be glycosylated, and may have a non-glycosylated peptide or hapten attached thereto. The non-glycosylated peptide is specifically recognized by the antibodies associated with the remover.
It is also preferred, for those therapeutic agents which must enter the cell in order to be effective, that they be linked to a peptide blocker which prevents the therapeutic agent from entering a cell. The blocker is preferably bound by the binding molecules.
The preferred means for linking such therapeutic agents with a blocker is with a hydrophobic, lipophillic peptide linker, such as that described in U.S. Pat. No. 5,149,782. There should also be a cleavage site between the blocker and the therapeutic agent, so that the blocker can be cleaved and allow the therapeutic agent to enter the cell.
The immunoconjugate of the therapeutic agent, the linker and the blocker should be small to minimize the phagocytosis by reticuloendothelial system (xe2x80x9cRESxe2x80x9d) cells. Preferbly, during the time the blocker is bound at the target site by the binding molecules, the blocker will be cleaved and released. The hydrophobic, lipohillic linker will tend to blend with the cell membrane, and thereby enhance the entry of the therapeutic agent into the cell.